Mycobacterium tuberculosis (TB) infects approximately one-third of the world's population, with approximately nine million new cases of TB occurring each year and two million deaths attributable to the disease. Culture based methods remain the gold standard for diagnosing drug resistant TB, but M. tuberculosis has a doubling time of 15 - 20 hours, which means that culture-based diagnosis (let alone drug-susceptibility testing) can require several weeks. The genetics of MDR-TB, however, poses a problem for PCR, molecular beacon, or line-probe tests, because the extent of known drug-resistance signatures greatly exceeds the multiplexing capacity of conventional PCR assays. At the same time, microarrays are still limited to the research community due to reagent and array expense, large and expensive analysis instruments, and labor and time intensive protocols that typically require an advanced academic degree to operate, use or interpret. What is therefore needed for MDR-TB diagnostics, especially in low-resource settings, is a highly multiplexed test (e.g. an array) with the sensitivity of PCR, configured as an entirely closed-amplicon test (akin to TaqMan(tm) PCR), with a technology footprint and price point that moves array-based molecular diagnostics towards low-resource settings where the disease is most prevalent. Akonni Biosystems has developed a prototype MDR-TB PCR TruArray(tm) and field-portable, low-cost analysis system to address this need. The objective of this project is to modestly expand test coverage for MDR- and XDR-TB genotyping, complete the manufacturing scale-up and reagent packaging studies for the resulting kit(s), and perform pre-clinical verification experiments on isolates and amended sputum samples at the Wadsworth Center Laboratory of Clinical Mycobacteriology prior to deployment in regions (U.S. border counties;South Africa) where TB is most prevalent. Underlying the technical aims is the administrative and management effort associated with developing the tests according to the FDA Quality System Regulation (21 CFR 820). Successful development and distribution of the MDR-TB array to collaborating, international reference centers will provide the necessary pre-clinical and real-world use data to support distribution of the test kit into global programs in tuberculosis surveillance and control, understand the true frequency of MDR-TB in under-represented populations, and enable prompt treatment of infected individuals with a correct regimen of front-line drugs. PUBLIC HEALTH RELEVANCE: The MDR- and XDR-TB PCR array will be the first-of-its-kind combination of PCR and microarray technology in a simple, inexpensive test for Mycobacterium tuberculosis disease surveillance and diagnosis. The underlying platform developed herein will likewise find broad application in many areas of infectious disease diagnostics.